Neonatal brain injury is an important cause of death and disability, with pathways of oxidant stress, inflammation, and excitotoxicity leading to damage that progress over a long period of time. Therapies have classically targeted individual pathways during early phases of injury, but more recent studies indicate that growth factors may also enhance cell proliferation, differentiation and migration of newly born neurons over time. Understanding how the cascade of injury responses occur and the key modulators during each phase will lead to more rationale therapies. Oxidative stress is a critical mediator of the injury response after neonatal hypoxia ischemia. We have shown that hydrogen peroxide toxicity is related to cell death but a paradox exists such that H2O2 may also be a signaling molecule that is involved in neuroprotection. In the previous cycle of this grant, to gain insight into potential new therapies we focused on hypoxic preconditioning to better understand the endogenous protective response that might occur after hypoxic-ischemic (HI) insults. We hypothesized that H2O2 serves as a critical signaling molecule for activation of hypoxia inducible factor 1 (HIF-1) t promote protection in the brain after neonatal ischemia. To address this hypothesis we have been examining the mechanisms by which HIF-1 is regulated by H2O2 to generate a cytoprotective response through the use of HIF conditional knockouts and glutathione peroxidase transgenics and knockouts. We now hypothesize that during mild HI, low levels of H2O2 upregulate HIF-1a and 2a in neurons and astrocytes respectively to enhance cell survival through paracrine signaling. Understanding the exact timing and control of this signaling is essential for translation of Epo dosing in the clinic. Therefore, this work will not only move the field forward in regard to understanding mechanisms, but provide a translatable therapy, Epo, to the bedside of asphyxiated term newborns. We will address this hypothesis in three specific aims. First, we will understand how the temporal and cell-specific regulation of H2O2 levels after HI in vivo leads to HIF upregulation and downstream production of Epo, secondly, we will understand if paracrine signaling is important for Epo production and cell survival after HI in vitro, and finally we will determine whether exogenous Epo will protect the brain from severe injury by overcoming the downregulation of HIF from high H2O2. The synergistic effects of hypothermia and Epo will also be assessed.